Means, standard deviations, and medians for variables and the results of the Mann–Whitney U-test by sex.

Means, standard deviations, and medians for variables and the results of the Mann–Whitney U-test by sex.</p

<i>Schizosaccharomyces pombe</i> Strains used in this Study.

<p><i>Schizosaccharomyces pombe</i> Strains used in this Study.</p

TOR inhibition induces the transfer of Agp3, Isp5, Aat1, and Put4 from trans-Golgi/endosomes into the vacuoles.

<p>(A) The co-localization of Agp3, Isp5, Aat1, and Put4 with Krp1, a marker for trans-Golgi/endosomes, in normal culture condition. Representative fluorescent images of the wild-type cells co-expressing Krp1-RFP and YFP-fused Agp3 (KP6624), Isp5 (KP6662), Aat1 (KP6665), or Put4 (KP6664) protein are shown. YFP and RFP signals are shown in green and red, respectively, and the merged signals are yellow. Arrowheads indicate the co-localization of Agp3-positive dots with the Krp1-positive structures. Scale bar, 10 μm. (B) The co-localization of Agp3 with Fnx1, a vacuole-enriched amino acid transporter, after Torin–1 treatment. Representative fluorescent images of the wild-type cells co-expressing Agp3-RFP and Fnx1-YFP (KP6644) with vehicle (Veh) or Torin–1 (Torin) treatment for 2 h. RFP and YFP signals are shown in red and green, respectively, and the merged signals are yellow. Scale bar, 10 μm.</p

Participants’ characteristics (<i>n</i> = 865).

Participants’ characteristics (n = 865).</p

Constitutive Tor2 activity is sufficient for Agp3 localization at trans-Golgi/endosomes.

<p>Representative images of Δ<i>tsc2</i> cells expressing Agp3-YFP with vehicle (Veh) or Torin–1 (Torin) treatment are shown. The cells were grown to the log phase in EMM, and subjected to drug treatment. In this experiment, Torin–1 treatment for 4 h rather than 2 h was required for it to exert its effect, which was perhaps due to the elevated Tor2 activity induced by <i>tsc2</i> deletion. YFP and FM4-64 signals are shown in green and red, respectively, and the merged signals are yellow. Scale bar, 10 μm.</p

Nitrogen depletion induces the transfer of Agp3 from trans-Golgi/endosomes to the plasma membrane, and then into the vacuoles.

<p>(A) The effect of nitrogen depletion on Agp3 localization. Representative images of wild-type cells expressing Agp3-YFP (KP6154) in nitrogen-rich medium (N-rich) or after the shift to nitrogen-depleted medium for 1 h, 4 h, and 9 h are shown. Scale bar, 10 μm. (B) The localization of Agp3 in FM4-64-stained vacuoles under nitrogen depletion for 9 h. Wild-type cells expressing Agp3-YFP were cultured in nitrogen-depleted medium for 8 h, and FM6-64 was loaded for 1 h in recycled nitrogen-depleted medium to avoid nutrient re-supplementation. Scale bar, 10 μm. (C) The expression of Agp3-YFP and its degradation by vacuolar proteases under nitrogen depletion. The wild-type cells (KP6154, WT, (+)) and Δ<i>isp6</i>Δ<i>psp3</i> cells (KP6635, Δ<i>isp6</i>Δ<i>psp3</i>, (+) expressing Agp3-YFP were collected without (0 h) or with nitrogen depletion (1 h, 4 h, and 9 h), and the proteins were extracted for western blotting. Agp3-YFP was detected with green fluorescent protein antibody. The wild-type cells without Agp3-YFP expression (KP5080, WT, (-)) were similarly analyzed as negative controls. The same immunoblot is shown with shorter and longer exposures. Tubulin expression was used as an internal control.</p

Systematic analysis of the localizations of several representative amino acid transporters with or without TOR inhibition in fission yeast.

<p>Representative fluorescent images of the wild-type (WT) cells expressing YFP-fused Agp3 (KP6154), Isp5 (KP6153), Aat1 (KP6156), Put4 (KP6155), Fnx1 (KP6157), Can1 (KP6158), or Fnx2 (KP6159) protein with vehicle (Veh) or Torin–1 (Torin) treatment for 2 h. The cells were grown to the log phase in EMM, and were subjected to vehicle or Torin–1 treatment. To visualize the vacuolar membranes, FM4-64 was loaded for 1 h before the Torin–1 treatment. YFP and FM4-64 signals are shown in green and red, respectively, and the merged signals are shown in yellow. Scale bar, 10 μm.</p

Increased Tor2 activity delays the transfer of Agp3 to the plasma membrane under nitrogen depletion.

<p>(A, B) The effect of constitutively active <i>tor2</i> mutants on the transfer of Agp3 to the plasma membrane under nitrogen depletion. Representative fluorescent images of wild-type cells (KP6154, WT), <i>tor2</i>^L1310P cells (KP6637), and <i>tor2</i>^E2221K cells (KP6638) expressing Agp3-YFP in nitrogen-rich medium (N-rich) or after a shift to nitrogen-depleted medium (N-depletion) for 10 min, 20 min, 30 min, and 60 min (A), and of the same cells of another batch under nitrogen depletion for 4 h (B). Scale bar, 10 μm. (C, D) The effect of <i>tsc2</i> deletion on the transfer of Agp3 to the plasma membrane under nitrogen depletion. Representative fluorescent images of the wild-type cells (KP6154, WT) and Δ<i>tsc2</i> cells (KP6434) expressing Agp3-YFP in nitrogen-rich medium (N-rich) or after the shift to nitrogen-depleted medium (N-depletion) for 1 h (C) and 4 h (D). Scale bar, 10 μm. (E) Rescue of the nitrogen depletion-induced transfer of Agp3 to the plasma membrane in Δ<i>tsc2</i> cells by the <i>tor2-287</i> mutation. Representative images of the wild-type cells (KP6154), Δ<i>tsc2</i> cells (KP6434), <i>tor2-287</i> cells (KP6244), and <i>tor2-287</i>Δ<i>tsc2</i> cells (KP6448) expressing Agp3-YFP under nitrogen depletion for 1 h are shown. Scale bar, 10 μm.</p

The Loss of Lam2 and Npr2-Npr3 Diminishes the Vacuolar Localization of Gtr1-Gtr2 and Disinhibits TORC1 Activity in Fission Yeast

<div><p>In mammalian cells, mTORC1 activity is regulated by Rag GTPases. It is thought that the Ragulator complex and the GATOR (GAP activity towards Rags) complex regulate RagA/B as its GDP/GTP exchange factor (GEF) and GTPase-activating protein (GAP), respectively. However, the functions of components in these complexes remain elusive. Using fission yeast as a model organism, here we found that the loss of Lam2 (SPBC1778.05c), a homolog of a Ragulator component LAMTOR2, as well as the loss of Gtr1 or Gtr2 phenocopies the loss of Npr2 or Npr3, homologs of GATOR components Nprl2 or Nprl3, respectively. These phenotypes were rescued by TORC1 inhibition using pharmacological or genetic means, and the loss of Lam2, Gtr1, Gtr2, Npr2 or Npr3 disinhibited TORC1 activity under nitrogen depletion, as measured by Rps6 phosphorylation. Consistently, overexpression of GDP-locked Gtr1^S20L or GTP-locked Gtr2^Q60L, which suppress TORC1 activity in budding yeast, rescued the growth defect of Δ<i>gtr1</i> cells or Δ<i>gtr2</i> cells, respectively, and the loss of Lam2, Npr2 or Npr3 similarly diminished the vacuolar localization and the protein levels of Gtr1 and Gtr2. Furthermore, Lam2 physically interacted with Npr2 and Gtr1. These findings suggest that Lam2 and Npr2-Npr3 function together as a tether for GDP-bound Gtr1 to the vacuolar membrane, thereby suppressing TORC1 activity for multiple cellular functions.</p></div

Lam2, Npr3 and Npr2 are necessary for maintaining the vacuolar localization and the protein levels of Gtr1 and Gtr2.

<p>(A, B) Intracellular localization of Gtr1-GFP (A) or Gtr2-GFP (B) in wild-type (wt) cells, Δ<i>lam2</i> cells, Δ<i>npr3</i> cells or Δ<i>npr2</i> cells are shown. The leucine auxotrophic wild-type (wt, HM123), Δ<i>lam2</i> cells (KP6607), Δ<i>npr3</i> cells (KP6551) and Δ<i>npr2</i> cells (KP6586) transformed with the plasmids expressing Gtr1-GFP (pKB8702) (A) or Gtr2-GFP (pKB9133) (B) under <i>nmt1</i> promoter were grown to early log phase in EMM medium at 27°C, and then were shifted to YES medium overnight. The fluorescent and DIC images were then acquired after the cells were treated with distilled water for 30 min. Scale bar, 10 μm. (C) The protein levels of Gtr1-GFP and Gtr2-GFP in wild-type (wt) cells, Δ<i>lam2</i> cells, Δ<i>npr3</i> cells and Δ<i>npr2</i> cells. The leucine auxotrophic wild-type (wt, HM123), Δ<i>lam2</i> cells (KP6607), <i>npr3</i>::<i>ura4</i>⁺ cells (KP6551), <i>npr2</i>::<i>ura4</i>⁺ cells (KP6586) and <i>npr2</i>::<i>KanMX</i>_<i>4</i> cells (KP6585) transformed the plasmids expressing, GFP (pKB2728), Gtr1-GFP (pKB8702) or Gtr2-GFP (pKB9133) under <i>nmt1</i> promoter were grown to early log phase in EMM medium without thiamine at 27°C for 20 h. Proteins were extracted and subjected to SDS-PAGE and immunoblot analyses with anti-GFP antibodies. A representative image of immunoblot analysis is shown in the upper panel. Signal intensities of the corresponding bands were quantified and normalized to the values of wild-type cells. The resultant normalized intensities were averaged across three independent blots, and are shown in the lower panel. ***<i>P</i><0.001 for Turkey’s test following one-way ANOVA for the comparisons with the value of wild-type cells.</p